The proposal centers on the study of in vitro model systems in which flow disturbances are created to mimic the shear stress gradients observed in vivo. These include areas of "flow separation" with recirculation, areas of "flow re-attachment" or stagnation, and areas of "flow recovery." Four different flow chambers will be prepared and evaluated. The first will be a parallel plate configuration with step protuberances that create flow separations and recirculation. The second will be a chamber with a 180-degree bifurcation, where a stagnation point is generated with large shear stress gradients to either side. The third is a radial flow chamber in which fluid enters through a central bore and moves radially away from this entrance point. In this chamber, a stagnation point is present at the center where fluid enters, and shear stress gradually dissipates as fluid moves away from the center, creating a "soft" shear stress gradient. Finally, inserting cover slips with endothelial cells into a flow chamber will create boundary layer flow. Cells positioned in these chambers will be evaluated for functional differences (depending on their location within the chamber) as a function of time after initiation of flow. To then relate the in vitro findings to in vivo characteristics of endothelial cells exposed to these types of flow characteristics, a cast of the rat vasculature will be made, from which shear stress gradients can be mapped, and functional studies for cells in vivo at equivalent locations will be made. It is proposed to examine patches of endothelial cells from regions of altered flow for proliferation (BrdU, PCNA), expression and localization of cell-cell adhesion molecules (VE-cadherin, occludin, PECAM), apoptosis and migration, in order to evaluate the impact of fluid flow disturbances on these endothelial cell characteristics. Gap junction composition will be studied by evaluating the location of connexins Cx43, 40, 37 in cells positioned at various points in the chambers using immunocytochemical techniques, and the expression of transcripts for these proteins will be sought using in situ hybridization techniques coupled with grain-counting, and antisense RNA amplification. Gap junction function will be studied by dye injection, as shown in the preliminary studies. Finally, in order to learn about the dynamic regulation of gap junction proteins (synthesis, degradation, assembly, and gating), it is proposed that nuclear run-off studies, pulse-chase labeling of RNA and protein, and phosphorylation of the gap junction proteins will be studied. In addition, cells transiently transfected with GFP-Cx43 will be used to study real-time re-localization of the protein depending on the shear gradient they are exposed to. Gap junction inhibition will be used to define whether the gap junctions are critical for the functional state of the endothelial cells (i.e. cell movement, proliferation, cell-cell adhesion and apoptosis).